1. Area of the Art
The invention relates generally to methods for targeting, enriching, detecting and/or isolating target nucleic acid sequence using RecA-like recombinase, and specifically to methods for targeting, enriching, detecting and/or isolating double-stranded nucleic acid target sequence using RecA-like recombinase in the presence of both a homologous probe and a heterologous probe.
2. Description of the Prior Art
A variety of recombinases, which catalyze in vitro homologous pairing and/or exchange of DNA strands, have been isolated from various prokaryotes and eukaryotes. Among these recombinases, RecA protein, a recombinase derived from Escherichia coli, has been extensively investigated. (Shibata T., Cell Technology, 9, No. 4, 281-291 (1990)). RecA protein is known to catalyze in vitro homologous pairing of single-stranded DNA with double-stranded DNA and thus to generate homologously paired triple-stranded DNA or other triple-stranded joint DNA molecules. (Rigas B., et al., Proc. Natl. Acad. Sci. USA, 83: 9591-9595 (1986); Hsieh P. et al., Proc. Natl. Acad. Sci. USA, 89: 6492-6496 (1992); Ferrin L. J. et al., Science, 254: 1494-1497 (1991), etc. ). RecA protein is also reported to catalyze the formation of a four-stranded DNA structure known as a double D-loop. In this reaction, two types of complimentary single-stranded DNA are used as homologous probes to target double-stranded DNA, which has a homologous site for the single-stranded DNA probe. (Sena E. P., Nature Genetics, 3: 365-372 (1993); Jayasena V. K. et al., J. Mol. Biol., 230:1015 (1993)). In addition to DNA-DNA hybridization, RecA protein can also promote RNA-DNA hybridization. For example, single-stranded DNA coated with RecA protein can recognize complimentarily with naked RNA (Kirkpatrick and Radding, 1992; Kirkpatrick et al., 1992).
By utilizing the property of RecA protein, methods have been developed for isolating specific double-stranded target DNA existing in a solution at a very low level (at a molar ratio of 1:50 molecules to 1: several hundred molecules) (Rigas B. et al., Proc. Natl. Acad. Sci. USA, 83: 9591-9595 (1986); Teintze M. et al., Biochem. Biophys. Res. Commun., 211: 804-811 (1995); U.S. Pat. No. 4,888,274). In situ hybridization methods have also been developed for detecting double-stranded target DNA in a fixed cell (WO 93/05177). These methods use RecA to mediate homologous paring between the target DNA and a homologous probe containing a sequence sufficiently complementary to the target DNA to form a homologous probe/target DNA complex.
Homologous pairing that is catalyzed by a recombinase, such as RecA protein, leads to the formation of networks (coaggregates) comprising RecA proteins, total DNA (target DNA+heterologous DNA) and homologous probes in the system (Tsang S. S. et al., Biochemistry, 24: 3226-3232 (1985); Gonda D. K. et al., J. Biol. Chem., 261: 13087-13096; Chow S. A. et al., Proc Natl. Acad. Sci. USA, 82: 5646-5650 (1985)). The efficiency of the homologous pairing occurring between double-stranded target DNA and single-stranded nucleic acid (homologous probe), which is complementary to the double-stranded target DNA and to which RecA protein is bound, is greatly reduced when an excessive amount of heterologous DNA is present in a given sample. To prevent this reduction in reaction efficiency, the amount of RecA protein, as well as that of RecA protein-coated homologous probe has to be increased in proportion to the amount of the total DNA in the sample (Rigas B. et al., Proc. Natl. Acad. Sci. USA, 83: 9591-9595 (1986)).
However, an increase in the amount of RecA protein-coated homologous probe in proportion to the total amount of DNA in a given sample increases the amount of the RecA protein/homologous probe/target DNA/heterologous DNA complex, which correspondingly increases the final amount of heterologous DNA contaminating the double-stranded target DNA recovered from the sample. Such contamination reduces the specificity of the reaction. Therefore, it seems a problem that in isolating double-stranded target DNA, the amount of the contaminating heterologous DNA recovered together with double-stranded target DNA is dependent on the amount of the homologous probe used in the reaction. This problem is especially significant if the ratio of target DNA to heterologous DNA is less than 1:1,000.
The heterologous DNA may be removed from the complex by utilizing the differences in sodium chloride or Mg2+ sensitivity between the probe/target DNA and the probe/heterologous DNA complexes (Honigberg S. M. et al., Proc. Natl. Acad. Sci. USA, 83: 9586-9590 (1986); Rigas B. et al., Proc. Nail. Acad. Sci. USA, 83: 9591-9595 (1986); U.S. Pat. No. 4,888,274). It was reported that when one uses a long, circular homologous probe of more than 5,000 nucleotides or a long stretch linear homologous probe of more than 3,000 nucleotides, it seems possible to isolate the double-stranded target DNA contained in a given sample at a ratio of 1 target DNA molecule per about 200 to 1,000 heterologous DNA molecules with certain specificity. The heterologous DNA may be removed from the complex of homologous probe/target DNA by utilizing the difference between the two complexes in sensitivity to sodium chloride or Mg2+. The stability of the homologous probe/heterologous DNA complex significantly differs from that of the homologous probe/target DNA complex.
However, when one uses a circular homologous probe containing a comparatively short sequence, e.g., a 700-nucleotide sequence complementary to a portion of the target DNA, the final yield of the double-stranded target DNA is significantly lowered. This is the case even if the concentration of the double-stranded target DNA is relatively high, such as one molecule per 50 molecules in a given sample. (Teintze M. et al., Biochem. Biophys. Res. Comm., 211: 804-811 (1995)). Therefore, when the amount of the double-stranded target DNA present in the DNA sample is extremely small (for example, at a molar ratio less than 1 molecule/1,000 molecules), it is unclear whether or not one can use a homologous probe to isolate double-stranded target DNA.
It is therefore extremely difficult to selectively eliminate the heterologous DNA from the RecA protein/homologous probe/target DNA/heterologous DNA complex, wherein the amount of heterologous DNA exceeding that of target DNA is more than 1,000-fold in a given sample. Particularly, when the length of the complementary sequence common to both the double-stranded target DNA and the homologous probe (the length of homologous probe) is short, i.e., less than 700 nucleotide sequence, the use of such short probes will make it more difficult for the selective elimination of heterologous DNA. This is because the bond within the homologous probe/target DNA complex, mediated by a short RecA-bound homologous probe, is significantly unstable in comparison with the bond within the same complex, mediated by a long homologous probe consisting of more than 3,000 complementary nucleotide sequence to the whole region of the double-stranded target DNA. Washing under stringent conditions will break the homologous probe/target DNA complex when a short probe is used.
When ATPxcex3S is employed as a co-factor, it is difficult to selectively eliminate only the heterologous DNA from the complex, since the bond within the RecA protein/homologous probe/heterologous DNA/target DNA complex is extremely stable. Alternatively, it is possible to eliminate RecA proteins from the complex prior to the process of removing the heterologous DNA from it. However, the stability of the homologous probe/target DNA complex without RecA protein distinctively decreases in comparison with that of the RecA protein/homologous probe/target DNA complex. This alternative, therefore, is not preferred. (Teintze M. et al., Biochem. Biophys. Res. Commun., 211: 804-811 (1995)).
Due to the fact that the amount of the contaminating heterologous DNA recovered with double-stranded target DNA is dependent on the amount of the homologous probe used in the reaction, as previously described, reducing the amount of homologous probe in the reaction may facilitate the efficiency of the selective elimination of heterologous DNA. Reducing only the amount of homologous probe, however, will also reduce the efficiency of homologous pairing between a homologous probe and a target DNA.
It has been demonstrated that the efficiency of the reaction, which depends on the length of the complementary sequence common to both the double-stranded target DNA and the homologous probe (the length of homologous probe), decreases as the length of the homologous probe deceases. (Hsieh P. et al., Proc. Natl. Acad. Sci. USA, 89: 6492 (1992); Sena E. P., Nature Genetics, 3: 365-372 (1993); Jayasena V. K. et al., J. Mol. Biol., 230:1015 (1993), etc.). Particularly, the efficiency of the homologous pairing is remarkably reduced when a homologous probe of less than several hundred nucleotide sequence is used in the reaction. Accordingly, it is extremely difficult to detect and/or isolate double-stranded target DNA efficiently by using a short homologous probe alone, particularly, when the amount of heterologous DNA existing in a given sample is more than 1,000-fold amount of target DNA.
It was reported that when one uses a long circular homologous probe of more than 5,000 nucleotides in homologous pairing between the homologous probe and a target DNA, contained at a ratio of one target DNA molecule to 1,000 heterologous DNA molecules in a sample, the reaction efficiency will be reduced when the amount of the RecA-coated homologous probe is reduced. The reduction of the reaction efficiency of homologous pairing, however, was suppressed by adding a long circular heterologous probe of more than 6,000 nucleotides to the pairing reaction at the ratio of less than sevenfold (molar ratio) of the amount of the homologous probe (Honigberg S. M. et al., Proc. Natl. Acad. Sci. USA, 83: 9586-9590 (1986)).
Therefore, a need exists to improve the efficiency and the specificity of the reaction of the conventional methods using a recombinase such as RecA protein for targeting, enriching, detecting and/or isolating an extremely small amount of double-stranded target DNA present in a given DNA sample (tar example, at a molar ratio of less than one molecule to 1,000 molecules) (M. Teintze et al, Biochem. Biophys. Res. Comm., 211, 804-811 (1995)). A need also exists for employing a short-strand homologous probe for various applications as discussed above.
It is therefore an object of the present invention to provide a method utilizing RecA-like recombinase for targeting, enriching, detecting, and/or isolating a target nucleic acid sequence with high efficiency and specificity. It is also an objective of the present invention to provide a RecA-like recombinase mediated method utilizing a short-strand probe for targeting., enriching, detecting, and/or isolating a low level target nucleic acid sequence in a given sample.
These and other objects and advantages are achieved by using a method of the present invention utilizing RecA-like recombinase for targeting, enriching, detecting and/or isolating a double-stranded nucleic acid target sequence in a sample. The method comprises the steps of providing a RecA-like recombinase, a homologous nucleic acid probe, a heterologous nucleic acid probe; and mixing the RecA-like recombinase, the homologous nucleic acid probe, the heterologous nucleic acid probe with the double-stranded target nucleic acid sequence in the sample, wherein the specificity of the homologous paring and/or strand exchange between the double-stranded nucleic acid target sequence and the homologous nucleic acid probe is increased due to the addition of the heterologous nucleic acid probe.
The methods in accordance with the present invention have been found to provide a number of advantages. As explained in greater detail below, it has been found that the present invention makes it possible to target, enrich, detect, and/or isolate a low-level target nucleic acid sequence contained in a given sample with high efficiency and specificity. The present invention also makes it possible to use a short homologous probe to detect a target sequence having only partial sequence been revealed.
The methods in accordance with the present invention are well suited for use in isolation and subsequent cloning of a target gene from a mixture of cDNAs or genomic DNAs. They are also well suited for use in screening of the target gene from various gene libraries, particularly, isolating and screening of the target gene whose nucleotide sequence or amino acid sequence is only partially revealed.
The methods of the present invention may also be used in the amplification of the target DNA sequence using RecA-like recombinase; mapping of various genes, such as RARE-based mapping using an oligonucleotide probe; nucleotide sequence specific modification or cleavage of the target DNA using an oligonucleotide.
Furthermore, the methods of the present invention may be used in in-situ hybridization methods using RecA-like recombinase to improve the specificity or the efficiency of the hybridization. They may also be used as a gene therapy technique by means of gene alternation or transcription inhibition using in vivo gene-targeting in living cells.
In this use of the method in accordance with the present invention, homologous pairing and/or strand exchange between a homologous probe and a target sequence, mediated by RecA-like recombinase, occurs in the presence of a RecA-like recombinase coated heterologous probe. In an embodiment of the present invention, the weight ratio of the amount of homologous probe to the amount of heterologous probe is between about 1:1 to 1:500.
The invention is defined in its fullest scope in the appended claims and is described below in its preferred embodiments.
The present invention is based on a surprise discovery that the addition of a heterologous probe to a RecA-like recombinase mediated reaction system increases the specificity of homologous pairing and/or strand exchange between a target DNA and a homologous probe complimentary to the target DNA, particularly, when a short homologous probe is used.
In addition, it is a surprise discovery of the present invention that reducing the amount of a homologous probe and adding a heterologous probe instead into a RecA-like recombinase mediated reaction system provide a higher specificity than that obtained when the homologous probe is used alone. This is also true even when a short linear homologous probe of less than several hundred nucleotides in length is used in the presence of at least 1,000-fold excess of heterologous DNA over the target DNA in a given sample.
Accordingly, the present invention provides a method for targeting, enriching detecting and/or isolating double-stranded nucleic acid target sequence in a sample by using RecA-like recombinase. The method comprises the steps of providing a RecA-like recombinase, a homologous nucleic acid probe, a heterologous nucleic acid probe; and mixing the RecA-like recombinase, the homologous nucleic acid probe, the heterologous nucleic acid probe with the double-stranded target nucleic acid sequence in the sample, wherein the specificity of the homologous pairing and/or strand exchange between the double-stranded target nucleic acid sequence and the homologous nucleic acid probe is increased due to the addition of the heterologous nucleic acid probe.
For the purpose of the present invention, the xe2x80x9cefficiencyxe2x80x9d is determined based on the amount of a target nucleic acid sequence that is targeted, enriched, detected, or isolated. The xe2x80x9cspecificityxe2x80x9d is determined based on the ratio of the amount of the target nucleic acid sequence that is targeted, enriched, detected, or isolated and the total amount of the target nucleic acid sequence plus the heterologous nucleic acid sequence that is targeted, enriched, detected, or isolated.
RecA-like recombinases utilized in the present invention include recombinases which have catalytic activity similar to of RecA protein derived from Escherichia coli. RecA protein can mediate both homologous pairing and/or strand exchange between appropriate DNA molecules in in vitro homologous recombination assays (Kowalczykowski,S., Ann. Rev. Biohpys. Biophysical Chem., 20:539-575 (1991), Radding C., M., Biochem. Biophys. Acta 1008:131-139 (1989), Radding C., M., J. Biol. Chem. 266:5355-5358 (1991); also see Golub, E., et al., Nucleic Acids Res. 20:3121-3125 (1992)). In addition to DNA-DNA hybridization, RecA protein can promote RNA-DNA hybridization. For example, RecA protein coated single stranded DNA can recognize complimentarity with naked RNA (Kirkpatrick, S. et al., Nucleic Acids Res. 20:4339-4346 (1992)). Therefore, any recombinase which can promote both homologous pairing and/or strand exchange between appropriate DNA molecules or between DNA and RNA molecules may be used in the present invention.
RecA-like recombinases have been isolated and purified from many prokaryotes and eukaryotes. The examples of such recombinases include, but are not limited to, the wild type RecA protein derived from Escherichia coli (Shibata T. et al., Method in Enzymology, 100:197 (1983)), and mutant types of the RecA protein (e.g., RecA 803: Madiraju M. et al., Proc. Natl. Acad. Sci. USA, 85: 6592 (1988); RecA 441(Kawashima H. et al., Mol. Gen. Genet., 193: 288 (1984), etc.); uvsX protein, a T4 phage-derived analogue of the protein (Yonesaki T. et al., Eur. J. Biochem., 148: 127 (1985)); RecA protein derived from Bacillus subtilis (Lovett C. M. et al., J. Biol. Chem., 260: 3305 (1985)); Recl protein derived from Ustilago (Kmiec E. B. et al., Cell, 29 :367 (1982)); RecA-like protein derived from heat-resistant bacteria (such as Thermus aquaticus or Thermus thermophilus ) (Angov E. et al., J. Bacteriol., 176: 1405 (1994); Kato R. et al., J. Biochem., 114: 926 (1993)); and RecA-like protein derived from yeast, mouse and human (Shinohara A. et al., Nature Genetics, 4: 239 (1993)).
In a preferred embodiment, RecA protein isolated and purified from a culture of Escherichia coli may be used. (E., Kuramitsu S. et al., J. Biochem., 90: 1033 (1981); Shibata T. et al., Methods in Enzymology, 100: 197 (1983)). Commercially available RecA protein may also be used (Boehringer-Mannheim, Pharmacia).
As used herein, the term xe2x80x9cnucleic acidxe2x80x9d encompasses RNA as well as DNA and cDNA. In the present invention, there is no specific restriction on a double-stranded nucleic acid target sequence for its length, type, or shape. For example, the double-stranded nucleic acid target sequence may be in a circular form or a linear form.
Preferably, the double-stranded nucleic acid target sequence is double-stranded target DNA. The double-stranded target DNA may be, but is not limited to, genomic DNA, cDNA derived from prokaryotes and eukaryotes, DNA derived from viruses or bacteriophages, fragments of such genomic DNA or cDNA, and various species of such DNA contained in various kinds of DNA libraries.
A double-stranded nucleic acid target sequence of the present invention may be a nucleic acid sequence contained in a solution; in fixed cells, cellular structures, and intercellular structures; or in living cells or cellular structures which are not fixed. Examples of cells, cellular structures and intercellular structures include, but are not limited to, bacteria, viruses, cellular organs such as nucleus, mitochondria, or chromosome, and parasites such as viruses or bacteria existing in biological samples such as blood. Cells or cellular structures may be fixed by means of conventional methods using organic solvents (e.g., methanol, ethanol etc.), acids (e.g., acetic acid) or cross-linking agents (e.g., formialin, paraformaldehyde). Those methods of fixing are well within the skill of the art in view of the instant disclosure.
A double-stranded nucleic acid target sequence of the present invention, if desired, may be labeled using conventional methods well known in the art. The target sequence may be labeled by, but not limited to, radio isotopes (e.g., 32P, 35S, etc.), fluorescent pigments (e.g., FITC, rhodamine etc.), enzyme labels (e.g., peroxidase, alkaline phosphatase etc.), chemiluminescent agents (e.g., acridinium ester etc.), and a various sorts of labels and ligands such as biotin or digoxigenin. Those labeling methods are well known to a person skilled in the art in view of the instant disclosure.
It should be understood that, although the methods in accordance with the present invention are primarily directed to the targeting, enriching, detecting, and/or isolating of double-stranded target DNA, they may also be used for other duplex nucleic acid target sequences. Examples of such duplex nucleic acid target sequences include, but are not limited to, DNA/RNA sequences or possibly double-stranded RNA elements, double-stranded or single-stranded nucleic acid sequences associated with viral, bacterial or parasitic pathogens.
As used herein, the term xe2x80x9csingle-stranded nucleic acid probexe2x80x9d means single-stranded nucleic acids. The term xe2x80x9cnucleic acidxe2x80x9d encompasses RNA as well as DNA and cDNA. Preferably, single-stranded DNA is employed. There is no restriction regarding the shape of a probe, and thus either a circular form or a linear form may be used.
Commercially available products of single-stranded or double-stranded nucleic acid sequences can be used as probes of the present invention. Probes can also be prepared using the methods well-known in the art. For example, probes may be prepared directly from viruses, bacteriophages, plasmids, cosmids, or other vectors which have a target sequence, by means of, e.g., a nick-translation method and random prime method or the like. If desired, a probe can be prepared by restriction digest of the segment corresponding to the probe from the vector and followed by electrophoretic isolation of the specific restriction fragment, or by amplification of the probe sequence using the PCR method. Although the probe thus obtained is usually double-stranded, if desired, a single-stranded probe can be obtained by denaturing or by sub-cloning of the double-stranded sequence into a single-stranded vector such as M13 phage. Alternatively, a single-stranded probe can be prepared by oligo-nucleotide synthetic methods. A long probe can be prepared by joining sub-fragments of the probe after the synthesis of those sub-fragments.
A homologous probe in accordance with the present invention is a nucleic acid probe which has a region of homology with a selected base sequence in a duplex nucleic acid target sequence. Preferably, a homologous probe is a single-stranded DNA probe. As used herein, the term probe xe2x80x9chomologyxe2x80x9d with the target means that the single-stranded probe and target duplex have a region of similar or exact base pair sequence which allows the probe to recognize and hybridize with the corresponding base pair region in the duplex target. The extent of base pair mismatching which is allowed without losing homology may be as high as 20% to 30%, depending on the distribution and lengths of mismatched base pairs. In a preferred embodiment, the homologous probe is single-stranded nucleic acid which contains the sequence having at least more than 70% homology to the partial or whole sequence of a target nucleic acid. In order to ensure sequence specific homologous pairing (hybridization reaction) between the double-stranded nucleic acid target sequence and the homologous probe, it is preferable that the homologous probe generally contains the sequence that is at least 90-95% homologous to the partial or whole sequence of the double-stranded nucleic acid target sequence.
A homologous probe in accordance with the present invention may be prepared by denaturing a double-stranded nucleic acid probe which is complementary to either one or both strands of a target sequence. The homologous probe may also contain an extended terminal portion which is not complementary to any of the nucleic acid strands in the sample. When both strands of a double-stranded homologous probe have such extended sequences at their termini, the extended sequences may be complementary to each other.
A homologous probe in accordance with the present invention is at least 15 nucleic acid residues in length, and preferably 15-2,000 nucleic acid residues in length. A longer polynucleotide probe (more than 2,000 nucleic acid in length) may also be used (Hsieh P. et al., Proc. Natl. Acad. Sci. USA, 89:6492 (1992)). Preferably, the length of required homology between a probe and a target is at least about 15 base pairs.
If desired, a homologous probe may be labeled for detection and/or isolation using conventional methods known in the art. Examples of the labels that may be used to label a homologous probe include, but are not limited to, radio isotopes (e.g., 32P, 35S etc.), fluorescent pigments (e.g., FITC, rhodamine etc.), enzyme labels (e.g., peroxidase, alkaline phosphatase etc.), chemiluminescent agents (e.g., acridinium ester etc.), and a various sorts of labels and ligands such as biotin or digoxigenin.
In the present invention, at least one species of the homologous probes is used in combination with at least one species of RecA-like recombinase. For example, a homologous probe may be coated with RecA-like recombinase to form a homologous probe/RecA-like recombinase complex using a method that is well known in the art. The amount of homologous probe to be used in a sample is usually 0.1-200 ng, preferably 0.5-150 ng, when the sample contains about 1 xcexcg of DNA (target DNA and heterologous DNA).
The heterologous probe employed in the present invention is a nucleic acid probe which is not sufficiently complementary to the target sequence. A heterologous probe is not sufficiently complementary to a target sequence if it cannot hybridize with the target sequence nor form a stable heterologous probe/target sequence complex. In a preferred embodiment, the sequence of the heterologous probe may show low complementarity to a sequence other than the target sequence in a target molecule comprising the target sequence and a vector sequence. For example, it may show low complementarity to a vector sequence, in which the target sequence is integrated or is used for constructing the gene library. However, it is preferred that such complementarity should be sufficiently low such that a heterologous probe/target molecule complex or a heterologous probe/vector sequence complex would not be formed.
The heterologous probe may be a DNA probe as well as an RNA probe. Preferably, the heterologous probe is single-stranded DNA. There is no restriction regarding the shape of a probe, and thus either a circular form or a linear form can be used. The probe may be prepared by denaturing the double-stranded heterologous probe.
For example, if the DNA sample is derived from a human, the heterologous probe used in the system may be a single-stranded nucleic acid probe derived from, but not limited to, a virus or bacteriophage, preferably, single-stranded phage DNA such as M13 or xc3x8 X174, or single-stranded DNA generated from the fragment of lambda phage DNA; a single-stranded nucleic acid probe derived from microorganisms, such as RNA derived from Escherichia coli or yeast; a single-stranded nucleic acid probe derived from eukaryotes except humans, such as single-stranded DNA generated from DNA fragments from salmon sperm.
Preferably, the heterologous probe employed in the present invention does not contain any labels or ligands.
The heterologous probe used in the present invention is at least 15 nucleic acid residues in length. Preferably it is about 15-20,000 nucleic acid residues in length, and more preferably 60-10,000 nucleic acid in length. A longer polynucleotide probe, such as a probe of more than 20,000 nucleic acid residues in length, may also be used.
In the present invention, at least one species of heterologous probes is used in combination with at least one species of recombinase. For example, a heterologous probe of the present invention may be coated with RecA-like recombinase to form a heterologous probe/RecA-like recombinase complex under reaction conditions well known in the art. The amount of a heterologous probe to be used in a sample is usually 0.1-500 ng, preferably 20-250 ng, when the sample contains about 1 xcexcg of DNA (target DNA and heterologous DNA).
In the present invention, co-factors may be added to the reaction system in association with the recombinase used. For example, when RecA protein derived from Escherichia coli is used as a recombinase, RecA protein can be bound to the homologous probe and the heterologous probe, according to the conventional method well known in the art, in the presence of co-factors. Examples of such co-factors include, but are not limited to, rATP, alone or in the presence of an rATP regeneration system, (for example, rATP regeneration system manufactured by Boehringer-Mannheim GmbH), ATPxcex3S, GTPxcex3S, dATP, a mixture of ATPxcex3S and ADP, a mixture of ADP and AlF4xe2x88x92 (aluminium nitrate and sodium fluoride), a mixture of dADP and AlF4xe2x88x92, a mixture of ATP and AlF4xe2x88x92, and a mixture of dATP and AlF4xe2x88x92 (Rigas B. et al., Proc. Natl. Acad. Sci. USA, 83: 9591-9595 (1986); Hsieh P. et al., Proc. Natl. Acad. Sci. USA, 89: 6492-6496 (1992); Ferrin L. J. et al., Science, 254: 1494-1497 (1991) Sena E. P., Nature Genetics, 3: 365-372 (1993): Teintze M. et al., Biochem. Biophys. Res. Commun., 211: 804-811 (1995); Jayasena V. K. et al., J. Mol. Biol., 230: 1015 (1993); Kowalczykowski S. (C. et al., Proc. Natl. Acad. Sci. USA, 92: 3478-3482 (1995); Moreus P. L. et al., J. Biol. Chem., 264: 2302-2306 (1989); U.S. Pat. No. 4,888,274, WO93/05177, WO93/05178, WO95/18236, WO93/22443 etc.). Preferably, co-factors are ATP (preferably, 1-5 mM), GTPxcex3S (preferably, 0.05-5 mM), ATPxcex3S (preferably, 0.01-3 mM), a mixture of ATPxcex3S and ADP (0.3-3 mM ATPxcex3S+) 0.3-1.1 mM ADP), or a mixture of ADP and AlF4xe2x88x92 [0.02-5 mM ADP+0.01-0.5 mM A1(NO3)3+5-50 mM NaF].
The homologous probe and the heterologous probe in accordance with the present invention may be used in either a single-stranded or a double-stranded form. In general, the probe is subjected to denaturation by heating for about 5 minutes at 95-100xc2x0 C. prior to the reaction, and after chilling the probe on ice for about 20 seconds to 1 minute, the probe is used for the binding reaction with RecA-like recombinase. If desired, the probe is centrifuged for about 1-10 seconds at 0-4xc2x0 C. prior to the binding reaction with the recombinase. Though the denatured single-stranded probe can be stored in a freezer at xe2x88x9220xc2x0 C., preferably, it is immediately mixed with the standard reaction solution containing a co-factor and a RecA-like recombinase in an ice water bath. The single-stranded probe is bound to the RecA-like recombinase by incubating the mixed solution at 37xc2x0 C. for 0-20 minutes (pre-incubation is not always necessary if ATP, GTPxcex3S, ATPxcex3S or a mixture of ATPxcex3S and ADP is used as a co-factor). In this reaction, RecA-like recombinase generally binds to the single-stranded probe at the ratio of one molecule to about 3 nucleotides of a single-stranded probe. The binding reaction can be carried out at the mixing ratio of the amount of RecA protein (monomer) to nucleotides of the probe ranging from 5:1 to 1:8, preferably from 3:1 to 1:6. If desired, the reaction is performed in the presence of single-strand DNA binding protein (SSB), topoisomeraseI or topoisomeraseII.
The homologous probe of the present invention may be used as a labeled homologous probe, when it is bound to RecA-like recombinase having various labels or ligands described in the reference WO95/18236, the text of which is incorporated herein by reference. On the contrary, it is not preferable to use the heterologous probe with RecA-like recombinase having various labels or ligands.
The homologous probe/RecA-like recombinase complex and the heterologous probe/RecA-like recombinase complex are added to the DNA sample under conditions for avoiding the denaturation of the double-stranded target DNA, for example, at a temperature lower than that for melting double-stranded DNA. The mixture is incubated at 37xc2x0 C. for generally 10 minutes-24 hours, preferably 15 minutes-2 hours to carry out homologous pairing (hybridization).
The ratio of the amount of homologous probe to that of the heterologous probe added to the reaction is generally 1:1-1:500, preferably 1:1-1:250. It is a surprise discovery of the present invention that the use of the heterologous probe together with the homologous probe in a ratio as described above greatly increases the specificity of targeting, enriching, detecting and isolating of a target nucleic acid sequence. As described in the Examples of the present invention, for example, the method of the present invention is applied to isolate double-stranded target DNA from a sample containing about 1 [g of total DNA with a molar ratio of target DNA to non-target DNA being 1:10,000. When 200 ng of RecA protein coated homologous probe of 60-275 nucleotide length is used alone, the specificity of the reaction is extremely low. A large amount of heterologous DNA other than target DNA is recovered in contamination with the double-stranded target DNA, though, a certain amount of the double-stranded target DNA is also recovered. When the amount of the RecA protein coated homologous probe is reduced to 50 ng and used alone, the efficiency of the reaction decreases in comparison with the efficiency of the reaction wherein 200 ng of homologous probe is used alone, and likewise the yield of the double-stranded target DNA decreases. However, when 50 ng of the RecA protein coated homologous probe is reacted with the sample DNA in the presence of 150 ng of the RecA protein coated heterologous probe, not only the reaction efficiency is increased and a larger amount of the double-stranded target DNA is recovered in comparison with the reaction wherein 200 ng of homologous probe is used alone, but also the specificity of the reaction greatly increases because of the strong inhibition of the recovery of heterologous DNA. When the amount of the homologous probe is further reduced (25 ng or 10 ng), and thus the ratio of the homologous probe to the heterologous probe is further reduced (homologous probe: heterologous probe=10:190, or homologous probe: heterologous probe=25:175), the specificity of the reaction is further distinctively improved.
It should be understood that the ratio of the amount of homologous probe to that of heterologous probe, also the ratio of RecA-like recombinase bound to the probes may be altered by considering the property of the sample and the like. In view the teaching of this disclosure, one skilled in the art can alter the ratio in light of the changes in the property of the sample without undue experiments.
In an embodiment of the present invention, the reaction solution for the homologous pairing (hybridization) may be prepared so that the final concentration of each component may be within the range as follows: 1-100 mM Tris-HC1 buffer or Tris-acetate buffer, 1-30 mM magnesium acetate or magnesium chloride, 0-50 mM sodium acetate or sodium chloride, 0-3 mM dithiothreitol, 0-100 mM EGTA, 0-50 mM spermidine, 0-10% glycerol, 1-5 mM ATP or 0.05-5 mM GTPxcex3S or 0.01-3 mM ATPxcex3S or 0.3-3 mM ATPxcex3S+0.3-1.1 mM ADP, 0.002-0.025 mM RecA protein, 0.5-150 ng of homologous probe and 20-200 ng of the heterologous probe per reaction, and about 1 lg of the DNA sample (target DNA+heterologous DNA).
The methods of the present invention may be used in connection with well-known methods in the art (U.S. Pat. No. 4,888,274, WO093/05177, WO093/05178, WO095/18236, Teintze M. et al., Biochem. Biophys. Res. Commun., 211: 804-811 (1995)) for detection or isolation of a complex formed between double-stranded target DNA and RecA protein coated homologous probe, wherein either the double-stranded target DNA, or RecA protein, or the homologous probe may be labeled with labels or ligands. For example, the complex may be a complex of homologous probe carrying various labels or ligands/RecA protein/double-stranded target DNA, or a complex of homologous probe/RecA protein carrying various labels or ligands/double-stranded target DNA, or a complex of homologous probe/RccA protein/double-stranded target DNA carrying various labels or ligands. In accordance with the present invention, those complexes are formed by homologous pairing (hybridization reaction) using RecA-like recombinase in the presence of both a homologous probe and a heterologous probe.
In a preferred embodiment, a method for isolating double-stranded target DNA comprises the steps of: labeling the homologous probe with biotin; coating the homologous probe and a non-labeled heterologous probe with RecA protein in the presence of an appropriate co-factor (for example, GTPxcex3S or a mixture of ATPxcex3S and ADP); reacting the RecA coated homologous and heterologous probes with a DNA sample containing double-stranded target DNA to form a complex of homologous probe/target DNA; and then capturing the complex on magnetic beads (BioMag, Dynal) conjugated with streptavidin. After washing out the DNA and probes which are not captured on the beads, biotin-labeled homologous probe/double-stranded target DNA complex bound to the beads is incubated in a solution containing NaCl at a temperature between the room temperature and 85xc2x0 C. for about 5-15 minutes to separate (elute) the double-stranded target DNA fraction from the beads. Preferably, the recovered target DNA fraction is then used to transform appropriate host cells, followed by selecting the transformed cells having the target DNA, and recovering the target DNA from the transformed cells.
To carry out the methods described above, the present invention also provides a kit for targeting, enriching, detection and/or isolation of a double-stranded target nucleic acid sequence in a sample. The kit may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the methods of the present invention.
For example, one of the container means may contain RecA-like recombinase. A second container may contain a solid phase designed to capture the complex of double-stranded target nucleic acid sequence and homologous probe labeled with markers or ligands. Containers are also provided for containing co-factors, heterologous probe and washing solution separately.
In an embodiment of the present invention, a kit in accordance with the present invention may comprise the following elements in separate compartments respectively: RecA-like recombinase, appropriate co-factors, a heterologous probe, a solid phase designed to capture the complex of double-stranded target DNA and homologous probe labeled with marker or ligand, and washing solution.
The method in accordance with the present invention may be used to target, enrich, detect and/or isolate with high specificity target nucleic acid molecules existing in a given sample at a very low level. In an embodiment of the present invention, the molar ratio of the target nucleic acid molecules to the non-target nucleic acid molecules may be less than 1:1,000. Preferably, the ratio may be more than 1:1,000,000, and more preferably more than 1:500,000.
The methods of the present invention are well suited for use in isolation and subsequent cloning of the target gene from a mixture of cDNAs or genomic DNAs as well as in screening for the target gene from various gene libraries. Examples of such gene libraries include, but are not limited to, DNA libraries, cosmid libraries, YAC libraries, and the like. Since a short strand probe may be used in the present invention, it becomes possible to isolate or screen a target gene whose nucleic acid sequence or amino acid sequence is only partially revealed.
The present invention may also be used in conjunction with well-known methods in the art for the following purposes: amplification of the target DNA sequence using RecA-like recombinase (U.S. Pat. No. 5,223,414, WO91/17267); mapping of various genes, such as RARE (RecA-Assisted Restriction Endonuclease cleavage) based mapping using an oligonucleotide probe (Ferrin L. J. et al., Nature Genetics, 6:379 (1994); Revet B. M. et al., J. Mol. Biol., 232:779 (1993)); nucleotide sequence-specific modification (methylation or alkylation) or cleavage of the target DNA using oligonucleotide (Koob M. et al., Nucleic Acid Res., 20:5831 (1992); Golub E. I. et al., Nucleic Acid Res., 20:3121 (1992); Mikhail A. et al., Biochemistry, 34:13098 (1995)). Applications of the present invention in conjunction with the above-known methods are well within the skill in the art in view of the instant disclosure.
Furthermore, the present invention may be used for isolation and/or detection of the specific target DNA of interest from clinical specimens containing a mixture of cDNA or genomic DNA. Therefore, the present invention allows the diagnosis of a variety of genetic aberration or mutation, or infectious diseases caused by a variety of pathogenic microorganism or viruses.
Moreover, the present invention may also be used in conjunction with an in-situ hybridization method, mediated by RecA-like recombinase (WO93/05177, WO095/18236), to improve the specificity or the efficiency of the hybridization. It is also applicable as a gene therapy technique and a transgenic technique to produce transgenic animals or plants by means of gene alternation or transcription inhibition using in vivo gene-targeting in the living cells (U.S. Pat. No. 5.468,629, WO093/2244; Golub E. I. et al., Nucleic Acid Res., 20: 3121 (1992); Golub E. I. et al., Proc. Natl. Acad. Sci. USA, 90; 7186 (1993), etc.). Applications of the present invention in conjunction with the above known methods are well within the skill in the art in view of the instant disclosure.
For example, when the present invention is used for in vivo gene-targeting in living cells, both RecA-like recombinase coated homologous probe and heterologous probe are used in forming the complex of RecA-like recombinase mediated homologous probe and double-stranded target DNA. Accordingly, a method for targeting a double-stranded nucleic acid target sequence in a sample of living cells by in vivo gene targeting method using a RecA-like recombinase may comprise the following steps: (a) providing a recombinase coated homologous probe and a recombinase coated heterologous probe; (b) introducing the homologous probe and the heterologous probe into the living cells; and (c) incubating said living cells for a sufficient period of time to allow the homologous probe to be transformed into the genome of the cells.
The conventional method of in vivo gene-targeting in living cells, which may be used in conjunction with the present invention, is described in U.S. Pat. No. 5,468,629 and WO093/2244, the relevant text of which is incorporated herein in its entirety by reference.